Cyclic peptidomimetic urokinase receptor antagonists

ABSTRACT

The present invention relates to cyclic peptides as inhibitors of urokinase binding to the urokinase receptor. Said cyclic peptides are suitable as pharmaceutical active substances for disorders mediated by urokinase and its receptor.

The present invention relates to cyclic peptides as inhibitors ofurokinase binding to the urokinase receptor, which are suitable aspharmaceutical active substances for disorders mediated by urokinase andits receptor. The substances of the invention are peptides derived fromthe uPA sequence and display, as ligands of the urokinase receptor(uPAR), an antagonistic action and are denoted uPAR antagonistshereinbelow.

The serine protease uPA (urokinase-type plasminogen activator) isresponsible for various physiological and pathological processes, suchas, for example, proteolytic degradation of extracellular matrixmaterial which is required for the invasiveness and migration of thecells and also for tissue remodeling. uPA binds with high affinity(K_(D)=10⁻¹⁰−10⁻⁹M) to the membrane-bound uPA receptor (uPAR) on thecell surface.

The binding of uPA to its receptor is involved in many invasivebiological processes such as, for example, metastasis of malignanttumors, implantation of trophoblasts, inflammations and angiogenesis.Therefore, uPAR antagonists are capable of inhibiting the invasiveness,metastasis and angiogenesis of tumors. uPAR antagonists may be employedas agents for the therapy of invasive and metastasizing cancers in whichuPA and uPAR appear at the invasive foci of tumors (Dano et al.: Thereceptor for urokinase plasminogen activator: Stromal cell involvementin extracellular proteolysis during cancer invasion, in: Proteolysis andProtein Turnover, Barrett, A. J. and Bond, J., HRSG, Portland Press,London, 1994, 239), for example in cancers of the breast, lung,intestine and ovaries. Moreover, uPAR antagonists may also be employedfor other purposes in which inhibition of the proteolytic activation ofplasminogen is required, for example for controlling disorders such asarthritis, inflammations, osteoporosis, retinopathies and forcontraception.

The uPA receptor is described in WO 90/12091 and also in thepublications Ploug et al., J. Biol. Chem. 268 (1993), 17539 and Ronne etal., J. Immunol. Methods 167 (1994), 91.

uPA is synthesized as single-chain molecule (pro-uPA) and convertedenzymically into an active two-chain uPA. The uPA molecule consists ofthree structurally independent domains, the N-terminally located growthfactor-like domain (GFD, uPA 1-46), a ring structural domain (uPA45-135) and the serine protease domain (uPA 159-141). GFD and the ringdomain together form the “amino-terminal” fragment of uPA (ATF, uPA1-135) which is generated by further proteolytic cleavage of two-chainuPA. ATF binds to the uPA receptor with a similar affinity as uPA.

The receptor-binding region of uPA extends across the region of aminoacids 12 to 32, since a peptide containing the uPA amino acid residues12 to 32 (with cysteine at position 19 being replaced by alanine).competes with ATF for binding to the uPA receptor (Appella et al., J.Biol. Chem. 161 (1987), 4437-4440). This study furthermore showed thatsaid peptide, even after cyclization due to bridging of the two cysteineresidues at positions 12 and 32, displayed an affinity for the uPAreceptor. In an alternative approach, Goodson et, al., (Proc. Natl.Acad. USA91 (1994), 7129-7133) identified antagonistic uPA peptides forthe uPAR by means of screening a bacteriophage peptide library. Thesepeptides showed no obvious sequence homology to the natural uPAR-bindinguPA sequence. More recent publications (Rettenberger et al., Biol. Chem.Hoppe-Seyler 376 (1995), 587-594); Magdolen et al., Eur. J. Biochem. 237(1996), 743-751; Goretzki et al., Fibrinolysis and Proteolysis 11(1997), 11-19) describe further studies on the uPAR binding region ofuPA. In this connection, the residues Cys19, Lys23, Tyr24, Phe25, Ile28,Trp30 and Cys31 were identified as important determinants for uPA/uPARinteraction. Said studies identified a uPA peptide with the uPA aminoacids 16 to 32 as the most active inhibitor.

Magdolen et al. (1996) supra analyze the uPAR binding region of the uPAmolecule by using a peptide with the uPA amino acids 14 to 32 andpeptides derived therefrom. However, these peptides and also peptidesused by other groups (cf. e.g. Appella et al. (1987) supra) have arelatively low affinity for UPAR.

WO-A-94/22646 discloses linear peptides having a length of 6 to 18 aminoacids which come from the region of the uPA amino acids 14 to 33. Thisstudy describes short peptides derived from uPA (uPA 21-29 and uPA21-26) being capable of influencing the growth of keratinocytes.Although WO-A-94/22646 indicates a possible use of the claimed peptidesfor blocking uPA/uPAR interaction, no data or indications of suchbinding studies whatsoever are shown. Moreover, the linear peptides uPA21-29 and uPA 21-26, denoted “preferred”, do not contain the minimaluPAR binding region of linear uPA peptides, which includes the sequenceregion of amino acids 19 to 31. Thus, the influence of said shortpeptides on keratinocyte growth is most probably not based on a uPA/uPARinteraction.

WO 98/46632 discloses uPAR peptide inhibitors which are derived from thelinear peptide uPA (19-31) and cyclic derivatives thereof and whichcarry D-amino acid residues in selected positions.

An example of a peptide inhibitor of this kind is the peptidecyclo(21,29](D-Cys²¹, Cys²⁹]uPA₂₁₋₃₀. This peptide already has quitehigh affinity for uPAR (IC₅₀=78 nM) which is only 4 times lower than theaffinity of the amino-terminal fragment of uPA (ATF=amino acids 1-135 ofurokinase) which has an IC₅₀ of 21 nM. The corresponding peptidecomposed exclusively of L-amino acids, cyclo[21,29]-[Cys²¹,Cys²⁹-uPA₂₁₋₃₀, has a hundred-fold lower activity compared with ATF.

It was the object of the present invention to modify the structure ofthe uPAR peptide inhibitor by incorporating isostructural or/andisofunctional natural and non-natural amino acids and thus to achievefurther improvement regarding the affinity for uPAR, serum stabilityor/and therapeutic action.

The present invention thus relates to compounds of the generalstructural formula (I):

wherein

-   -   X²¹ —X³⁰ are monomeric building blocks, preferably        aminocarboxylic acid residues and are derived from a structure        in which X²¹=D-Cys, X²²=Asn, X²³=Lys, X²⁴=Tyr, X²⁵=Phe, X²⁶=Ser,        X²⁷=Asn, X²⁸=Ile, X²⁹=Cys and X³⁰=Trp,    -   Y is a spacer and m is 0 or 1, and the monomeric building blocks        are linked via —CONR¹ or —NR¹CO bonds, in which R¹ in each case        independently is hydrogen, methyl or ethyl, and to        pharmaceutically acceptable salts and derivatives, thereof,        with the proviso that at least one of the amino acid residues        X²¹—X³⁰ of the lead structure is replaced by one of the amino        acid residues listed below:    -   X²¹: Asp, Glu, 2,3-diaminopropionic acid (Dap),        2,4-diaminobutyric acid (Dab), penicillamine (Pen), D-Pen,        allylglycine (Alg), ornithine (Orn), Lys,;    -   X²²: Gln, Asp, Glu;    -   X²³: Orn, Dap, Arg, His, citrulline (Cit), homocitrulline (Hci),        norleucine (Nle);    -   X²⁴: Phe, homophenylalanine (Hph),        1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic),        thienylalanine (Thi), Trp, phenylglycin (Phg), 1-naphthylalanine        (1-Nal), 2-naphthylalanine (2-Nal), Cha (cyclohexylalanine);    -   X²⁵: Tyr, Trp, Tic, Thi, Hph, Phg;    -   X²⁶: Thr, Val, homoserine (Hse);    -   X²⁷: Gln, Asp, Glu;    -   X²⁸: Val, Leu, 2-aminobutyric acid (Abu), tert-leucine (Tle),        norvaline (Nva), Nle, α-aminoisobutyric acid (Aib), Cha;    -   X²⁹: Asp, Glu, Dap, Dab, Alg, Pen, D-Pen, Orn, Lys;    -   X³⁰: Thi, Phe, Tyr, 2-Nal, 1-Nal, octahydroindolyl-2-carboxylic        acid (Oic), His, thiazolylalanine (Thia), Phg, tryptamine,        tryptophanamide (Trp-NH₂).

Preference is given to peptides in which at least one of the amino acidresidues X²¹—X³⁰ of the lead structure has one of the meanings listedbelow:

-   -   X²¹: D-Pen;    -   X²²: Gln;    -   X²³: Orn, Dap, Dab, Arg, Cit, Hci, Nle, His;    -   X²⁴: Phe, Thi, Hph, Phg, 1-Nal, 2-Nal, Cha;    -   X²⁵: Thi;    -   X²⁷: Asp;    -   X²⁸: Nle, Val, Cha;    -   X²⁹: Pen;    -   X³⁰: Phe, Thi, Tyr, Oic, 1-Nal, Hph, Thia, Trp-NH₂.

Particular preference is given to peptides in which at least one of theamino acid residues X²¹—X³⁰ of the lead structure has one of themeanings listed below:

-   -   X²¹: D-Pen;    -   X²³: Arg, Nle, Cit, Hci;    -   X²⁴: Phe, 1-Nal, 2-Nal, Cha;    -   X²⁵: Thi;    -   X²⁸: Nle, Cha;    -   X²⁹: Pen;    -   X³⁰: Trp-NH₂.

Y is a spacer group, for example a peptidic spacer group composed of oneor more amino acids, for example poly-Lys, or another spacer group, forexample a polyethylene glycol group. The peptide may be coupled tosupport substances via the group Y.

The peptides of the invention are cyclic peptides having a nine-memberedring, where at least 2, preferably at least 3 and particularlypreferably at least 4, of the amino acid residues forming the ring havea sequence from the uPA region 22 to 28.

The present invention further relates to compounds of the generalstructural formula (I):

wherein

-   -   X²¹—X³⁰ are monomeric building blocks, preferably        aminocarboxylic acid residues and are derived from a structure        in which X²¹=D-Cys, X²²=Asn, X²³=Lys, X²⁴=Tyr, X²⁵=Phe, X²⁶Ser,        X²⁷=Asn, X²⁸=Ile, X²⁹=Cys and X³⁰=Trp,    -   Y is a spacer and m is 0 or 1, and        the monomeric building blocks are linked via —CONR¹ or —NR¹CO        bonds, in which R¹ in each case independently is hydrogen,        methyl or ethyl, and to pharmaceutically acceptable salts and        derivatives thereof,        with the proviso that at least one of the amino acid residues        X²¹—X³⁰ of the lead structure is replaced by a non-proteinogenic        amino acid residue, with the resulting compounds preferably        having increased protease stability, in particular increased        stability against physiological proteases, for example proteases        present in blood or tissue, such as, for example, plasmin,        or/and proteases present in the digestive tract such as, for        example, pepsin, trypsin or chymotrypsin, compared with the lead        structure. Preferably, at least the amino acid residue Lys²³ is        replaced by a non-proteinogenic amino acid, i.e. by a        non-genetically encoded amino acid such as, for example, Orn,        Dap, Dab, Cit, Hci or Nle.

Suitable uPAR antagonists are, apart from peptides of the structuralformula (I), also pharmaceutically acceptable salts and derivativesthereof. Suitable derivatives are in particular those compounds whichhave modified reactive side chain groups or/and modified N- orC-terminal groups, for example amino or carboxyl groups. Examples ofsuch modifications are acylation, for example acetylation of aminogroups, or/and amidation or esterification of carboxyl groups, forexample amidation of the C-terminal amino acid. The monomeric buildingblocks are linked via NR¹CO or CONR¹ carboxamide bonds, i.e. thedirection of the peptide sequence can be reversed (retropeptides). R¹may be hydrogen, as in native polypeptides. On the other hand, however,R¹ may also be an alkyl radical, for example methyl or ethyl, and inparticular methyl, since N-alkylation of the amide bond can often have astrong effect on the activity (cf. e.g. Levian-Teitelbaum et al.,Biopolymers 28 (1989), 51-64). Unless stated otherwise, theα-aminocarboxylic acids are employed as monomeric building blocks in theform of L-enantiomers.

The peptides of the invention are cyclic compounds, with the monomericbuilding blocks X²¹ and X²⁹ being bridged with one another. Saidbridging may take place, for example, via the side chains of theparticular α-aminocarboxylic acid residues and bridging via disulfidebonds, for example between two cysteine residues, is particularlypreferred. However, other types of cyclization between amino acid sidechains are also possible, for example amide bonds between an amino acidwith an amino side group, for example ornithine or Lys, and an aminoacid with a carboxyl side chain such as, for example, Asp or Glu.Furthermore, the disulfide bridge may also be replaced by an alkylenebridge, in order to increase chemical stability. Moreover, linkages ofan amino acid side chain to the peptide backbone, for example linkage ofan amino side group, for example an ω-amino side chain, to theC-terminal end, and linkage of a carboxyl side group to the N-terminalend are also possible. A linkage of N- and C-terminus is also possible.The peptides of the invention are obtainable by chemical synthesis, asillustrated in the examples.

Furthermore, the present invention relates to a pharmaceuticalcomposition which contains as active substance at least one peptide orpolypeptide as defined above, where appropriate together withpharmaceutically common carriers, excipients or diluents. The peptidesor polypeptides of the invention are in particular used for preparinguPAR antagonists which are also suitable for controlling of disordersassociated with uPAR expression, in particular for controlling tumors.Furthermore, the peptides of the invention, as well as the leadstructure cyclo[21,29][D-Cys²¹,Cys²⁹]-uPA₂₁₋₃₀, can be employed asinhibitors of angiogenesis.

The present invention further relates to the use of the uPAR peptideantagonists of the invention for preparing targeting vehicles, forexample liposomes, viral vectors, etc., for cells expressing uPAR.Targeting may be carried out for diagnostic applications for controlledtransport of labeling groups, for example radioactive or nonradioactivelabeling groups.

On the other hand, targeting may be carried out for therapeuticapplications, for example for transporting pharmaceutical activesubstances, for example also for transporting nucleic acids for genetherapy.

The pharmaceutical compositions of the invention may be present in anyform, for example as tablets, as coated tablets or in the form ofsolutions or suspensions in aqueous or nonaqueous solvents. The peptidesare administered preferably orally or parenterally in liquid or solidform. The preferred carrier medium used for administration in liquidform is water which, where appropriate, contains stabilizers,solubilizers or/and buffers commonly used for injection solutions. Suchadditives are, for example, tartrate or borate buffer, ethanol, dimethylsulfoxide, complexers such as, for example, EDTA, polymers such as, forexample, liquid polyethylene oxide, etc.

Examples of solid carrier substances which may be employed foradministration in solid form are starch, lactose, mannitol,methylcellulose, talc, highly dispersed silicon oxide, high molecularweight fatty acids such as, for example, stearic acid, gelatin, agar,calcium phosphate, magnesium stearate, animal and plant fats or solid,high molecular weight polymers such as, for example, polyethyleneglycols. Furthermore, the formulations for oral application may alsocontain flavorings and sweeteners, if desired.

The therapeutic compositions of the invention may also be present in theform of complexes, for example with cyclodextrins such as, for example,γ-cyclodextrin. The dose administered depends on the age, state ofhealth and weight of the patient, on the type and seriousness of thedisease, on the type of treatment, on the frequency of administrationand the type of desired action. The daily dose of the active compound isusually 0.1 to 50 mg/kilogram of body weight. Typically, 0.5 to 40 andpreferably 1.0 to 20 mg/kg/day in one or more doses are sufficient inorder to achieve the desired effects.

The examples and figures described below are intended to furtherillustrate the invention.

In this connection,

FIG. 1 shows the comparison of the affinities of peptidescyclo[21,29][Cys^(21,29)]-uPA₂₁₋₃₀ andcyclo[21-29][D-Cys²¹,Cys²⁹]-uPa₂₁₋₃₀ (a) andcyclo[21,29][Cys^(21,29)]uPa₂₁₋₃₀ and the corresponding peptide amide(b), respectively;

FIG. 2 shows IC₅₀ values of modifications of the lead structurecyclo[21,29][D-Cys²¹,Cys²⁹]uPA₂₁₋₃₀;

FIG. 3 shows a diagrammatic representation of preferred lead structuremodifications;

FIG. 4 shows the stability of the peptides cyclo[19,31]-uPA₁₆₋₃₂,cyclo[21,29][D-Cys²¹, Tic²⁵, Cys²⁹]-uPA₂₁₋₃₀ andcyclo[21,29]-[D-Cys²¹,Cys²⁹]uPA₂₁₋₃₀ in human serum (a) and heparinizedhuman blood (b);

FIG. 5 shows the plasmin resistance of uPA peptides after substitutionof Lys²³ by non-proteinogenic amino acids.

EXAMPLES

1. Methods

1.1 Solid Phase Peptide Synthesis

Linear peptides were synthesized on a 2-chlorotrityl resin; (Barlos etal., Int. J. Pept. Protein Res. 37 (1991), 513 to 520) or a tritylchloride polystyrene resin using an Applied Biosystems Model 431 Apeptide synthesizer or a multiple peptide synthesizer Model Syro II(MultiSynTech). Applying the orthogonal Fmoc strategy (Carpino and Han,J. Org. Chem. 37 (1972), 3404-3409; Fields and Noble, Int. J. PeptideProtein Res. 35 (1990), 161-214), the amino acid side chains wereblocked with the protective groups trityl (Asn, Cys, Gln and His),tert-butyloxycarbonyl (Lys and Trp), tert-butyl (Asp, Glu, Ser, Thr andTyr), acetamidomethyl (Cys) and 2,2,5,7 8-pentamethylchroman-6-sulfonylor 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Arg). Coupling wascarried out in dimethylformamide at room temperature using a three-foldexcess of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate/1-hydroxybenzo-triazole/Fmoc amino acid with 2.5equivalents of N-ethyldiisopropylamine in N-methylpyrrolidone. The Fmocgroup was removed by sequentially treating the resins with an excess of40% and 20%, respectively, piperidine in dimethylformamide. The peptideswere cleaved off and the protective groups of the side chains wereremoved at the same time by treatment with 93% trifluoroacetic acid/5%triisopropylsilane/3% H₂O (0° C./1 h; room temperature/1 h). In the caseof 2,2,5,5,7,8-pentamethylchroman-6-sulfonyl-protected Arg groups, thepeptides were incubated at room temperature for an additional 12 h. Thecrude peptides were precipitated with diethyl ether at −30° C.,dissolved in methanol, precipitated as before, dissolved in tert-butanoland lyophilized. Tryptophan-containing peptides were additionallytreated with 5% acetic acid for 2 h prior tolyophilization.

The peptides were purified by HPLC using a reverse phase C-18 column(Nucleosil 1005-C18) or a YMC-Pack ODS column. The cyclization wascarried out by forming a disulfide bridge. The oxidation required forthis was carried out by taking up 0.1 to 0.3 mg/ml purified linearpeptides in 80% water and 20% DMSO (v/v) and removing the solvent underreduced pressure after 10 h. The cyclic peptides were then againpurified by HPLC as described before.

1.2 Mass Spectrometry and Amino Acid Analysis

The purified and desalted peptides were analyzed on the HPLC system 140B (Applied Biosystems, Foster City, USA). UV absorption was measured at206 nm using UVIS 200 (Linear Instruments, Reno, USA) detector. Thechromatography was carried out on an Aquapore 3 μ (Applied Biosystems,Foster City, USA) reverse phase column (1 mm×50 mm) with a flow rate of20 μl/min. The solvent system was 0.1% TFA. in water. (A) and 0.1% TFAin acetonitrile (B). The HPLC system was coupled to an atmosphericpressure ionization source which was connected to an API III tandemquadrupole instrument (Sciex, Perkin Elmer, Thornhill, Canada).

The quadrupole m/z scale was calibrated using the ammonium adducts ofpolypropylene glycol. The average masses were calculated from the m/zpeaks in the charge distribution profiles of the multiply charged ions(Covey et al., Rapid Commun. Mass Spectrom. 2 (1988), 249-256; Fenn etal., Science 246 (1989), 64-71).

The amino acid analysis was carried out according to the ninhydrinmethod using the analysis system 6300 (Beckman Instruments, Fullerton,USA), after hydrolyzing the peptides by the TFA-HCl vapor-phase method(Tsugita et al., J. Biochem. 102 (1987), 1593-1597), which allowsquantitative determination of the peptide concentration.

1.3 Flow Cytometry

The capacity of the synthetic peptides for inhibiting uPA/uPARinteraction was determined using the human promyeloid cell line U937 assource for cellular native uPAR by means of flow cytometry on a FACScanflow cytometer (Becton-Dickinson, Heidelberg, Germany) (Chuchulowski etal., Fibrinolysis 6, Suppl. 4 (1992), 95-102; Magdolen et al., (1996),supra). The U937 cells were stimulated with 1 mM phorbol 12-myristate13-acetate (PMA) for 48 h. After stimulation with PMA, the U937 cellsexpress considerable amounts of cell surface-associated uPAR.

The stimulated cells were treated with 50 m glycine-HCl, 0.1 NaCl, pH3.6 at room temperature for 1 min, in order to dissociate endogenousreceptor-bound uPA. Subsequently, the acidic buffer was neutralized by0.5 M HEPES, 100 mM NaCl, pH 7.5. The cells were then immediately washedtwice with PBS/0.1% bovine serum albumin (BSA) and centrifuged at 300×gat room temperature for 10 min. The cells were resuspended in PBS/0.1%BSA, adjusted to a concentration of 10⁶ cells per ml and incubatedsimultaneously with 1.6 ng of FITC-conjugated pro-uPA and differentamounts of the synthetic peptides at room temperature for 45 min. Priorto analysis, propidium iodide, a fluorescent dye binding specificallydouble-stranded DNA, was added to each sample, in order to determine theviability of the analyzed U937 cells. Damaged, propidium iodide-labeledcells were excluded from the analysis.

1.4 Solid Phase uPAR/uPA Binding Test

In addition to the analyses by flow cytometry, a solid phase ATF ligandbinding test was carried out to determine the interactions of syntheticpeptides with uPAR. For this, microtiter plates were coated withrecombinant human uPAR from CHO cells (Wilhelm et al., FEBS Lett. 337(1994), 131-134; Magdolen et. al., Elektrophoresis 16 (1995), 813-816)and the remaining protein-binding sites were saturated with 2% BSA(w/v). After incubation with the samples (0.6 ng of ATF together with 15μg of synthetic peptide per ml) and two or more washing steps, theamount of ATF which had bound to uPAR immobilized on the microtiterplate was determined using a biotinylated monoclonal mouse antibodyagainst the ATF ring domain (No. 377, American Diagnostica, Greenwich,Conn., USA) and subsequently adding avidine-peroxidase conjugate and3,3′,5,5′-tetramethylbenzidine/H₂O₂ as peroxidase substrate. Thepresence of synthetic peptides competing with ATF binding to uPARreduces conversion of the chromogenic substrate.

1.5 Determination of Peptide Stability in Body Fluids

The stability of uPA-derived peptides in human serum or complete bloodwas assayed in vitro.

Human sera were prepared by allowing venous blood to coagulate withoutanti-coagulant in polypropylene tubes at 37° C. for 45 min. The clotsadhering to the vessel wall were removed using a plastic stick andremoved by centrifugation at 1200×g at room temperature for 12 min. Theserum in the supernatant was removed and either used freshly forstability studies or frozen in aliquots at −20° C. for further use.

For stability studies in human complete blood, coagulation of venousblood was inhibited by heparin sodium (1 000 IU per 10 ml of completeblood), and the blood was used freshly. The peptides to be tested wereadded to the body fluids in the form of peptide-mixture stock solutions(≧1 μg per peptide in H₂O) at a concentration of 5 μg/100 μl of serum or200 μl of complete blood (per peptide) and incubated at 37° C. forvarious times. Prior to HPLC analysis, complete blood incubations werecentrifuged at 16 000×g at room temperature for 5 min and the plasma inthe supernatant was removed.

Sera and plasmas were prepurified via HLB precolumns (Waters GmbH, OasisHLB extraction cartridge 1 cm³/30 mg) prior to HPLC analysis. For thispurpose, 100 μl of liquid were diluted with PBS to 1 ml and applied tosaid HLB precolumns equilibrated with 1 ml of 100% methanol and 1 ml ofH₂O. The columns were washed with 1ml of 5% methanol in H₂O and elutedwith 1 ml of 100% methanol. The first 200 μl (4 drops) of the eluatewere discarded as void volume. The next 500 μl of eluate were dilutedwith 500 μl of PBS and analyzed by HPLC. The remaining 300 μl of theeluate were discarded. HPLC analysis was carried out using a YMC-5 C₁₈analytical column and a 20-60% gradient of H₂O, 0.1% trifluoroaceticacid and acetonitrile, 0.1% trifluoroacetic acid over 30 min anddetecting the analytes at 220 nm.

1.6 Determination of Protease Resistance

The sensitivity of peptides against the attack of various proteases ofthe digestive tract was determined in vitro using purified enzymes undersuitable buffer conditions. In general, 10 μg of peptide were incubatedin a 100 μl volume with 2 μg of protease at 37° C. for 30 min under thebuffer conditions for the activity controls as stated by themanufacturer. After incubation and dilution with 300 μl of H₂O, themixtures were analyzed directly, without prepurification via aprecolumn, by HPLC using a 20-60% gradient of H₂O, 0.1% trifluoroaceticacid and acetonitrile, 0.1% trifluoroacetic acid.

Pepsin (Sigma, Deisenhofen, Germany) was incubated with peptides in 52mM HCl. Incubations with trypsin (Sigma) were carried out in 63 mMsodium phosphate pH 7.6. Chymotrypsins α, β, γ,δ (ICN) were incubated in50 mM CaCl₂ and 40 mM Tris/HCl pH 7.8. This was followed by incubatingwith bacteriae proteinase K in unbuffered water. The positive controlused was the *peptide cyclo[19,31]-uPA₁₆₋₃₂ which corresponds to theoriginal sequence of the uPA omega loop except for the cysteine bridge.The peptide sensitivity against the tissue protease plasmin (Sigma) wasassayed in mixtures with 10 or 5 μg of peptides and 0.05 U of plasmin in100 μl of 200 mM sodium phosphate pH 7.5 at 37° C. and a 30 minincubation period.

2. Results

2.1 Inhibitory Action of the Peptide Cyclo[21,29][D-Cys²¹,Cys²⁹]uPA₂₁₋₃₀

FIG. 1 a depicts the inhibitory action of the peptidecyclo[21,29][D-Cys²¹,Cys²⁹]uPA₂₁₋₃₀ compared with the cyclic peptidecyclo[21,29][Cys^(21,29)]-uPA₂₁₋₃₀ which consists exclusively of L-aminoacids. The IC₅₀ of the cyclic peptide with D-Cys at position 21 wasdetermined to 78 nM, while the IC₅₀ of the cyclic peptide composed ofonly L-amino acids was determined to 2 260 nM. In comparison therewith,the IC₅₀ of the amino-terminal fragment of uPA (uPA amino acids 1 to135) is 21 nM.

2.2 Synthesis of Modified uPA Peptides

Using cyclo[21,29][D-Cys²¹,Cys²⁹]uPA₂₁₋₃₀ as lead structure, furthercyclic peptides were prepared, in which particular amino acids weresubstituted by other, in particular non-proteinogenic amino acids. Therelative activities compared with the lead structure are depicted inFIGS. 1 b and 2.

FIG. 3 depicts examples of particularly preferred modifications of thelead structure.

2.3 Results of the Studies on Peptide Stability in Human Serum andComplete Blood

The stability of peptides cyclo[19,31]-uPA₁₆₋₃₂ (A),cyclo[21,29][D-Cys²¹,Tic²⁵,Cys²⁹]-uPA₂₁₋₃₀ (B) andcyclo[21,29][D-Cys²¹,Cys²⁹]-uPA₂₁₋₃₀ (C) in human serum and heparinizedhuman blood was assayed. FIGS. 4 a and 4 b depict the results of theseexperiments. The peaks at 20.5 min correspond to peptide A, the peaks at23.0 min correspond to peptide B and the peaks at 24.9 min correspond topeptide C.

The stability of the peptides inhuman serum (FIG. 4 a) was studied byadding a mixture of in each case 12.5 μg of the peptides to about 250 μlof serum. 100 μl thereof were diluted with PBS to 1 ml, prepurified onan HLB precolumn and analyzed immediately by HPLC (middle profile). Afurther 100 μl were analyzed after 20.5 h of incubation at 37° C.(bottom profile). As a control, in each case 5 μg of the peptides wereadmixed with 1 ml of PBS and analyzed (top profile). The peak at 21.2min corresponds to a nonidentified metabolite.

The stability in heparinized human blood (FIG. 4 b) was studied byadding 750 μl of freshly prepared heparinized human blood to 37.5 μg ofthe peptides. Immediately thereafter, 375 μl were removed bycentrifugation. 100 μl of the plasma supernatant were analyzed (middleprofile). The remaining 375 μl were incubated with gentle agitation at37° C. for 20.5 h and then analyzed (bottom profile). As a control, 5 μgof the peptides had already been admixed with 1 ml of PBS and analyzed(top profile).

After incubation in human serum at 37° C. for 20.5 h, peptide A(cyclo[19,31]-uPA₁₆₋₃₂) could no longer be detected. Instead a new peakwith relatively longer retention time (FIG. 4 a, bottom HPLC profile, RT21.233 min) appeared, which corresponds presumably to a metabolite of A.In contrast, the relative retention times and peak integrals of peptidesB (cyclo[21,29][D-Cys²¹Tic⁴⁵Cys²⁹]-uPa₂₁₋₃₀) and C(cyclo[21,29][D-Cys²¹Cys²⁹]-uPA₂₁₋₃₀) remained nearly the same, afterexposure to human serum. This allows the conclusion to be reached thatthe chemical identity and concentration of the two peptides areunaltered after a 20.5-hour exposure to human serum.

Exposing the peptides to freshly isolated heparinized complete bloodover 20.5 h, too, showed the instability of A. In this case, it was notpossible to detect either the unaltered substance or a suspectedmetabolite. In contrast, the peaks of peptides B and C appeared stablein human blood. Compared with the PBS control, the A peak appearsalready substantially reduced in the sample worked up and analyzedimmediately after addition to complete blood. Apparently, the shortperiod between addition of the peptides and prepurification onprecolumns (10-15 min) was sufficient to degrade a substantial part ofthe amount of peptide added (>80%).

The peak integrals of the two D-Cys derivatives B and C from plasma aredistinctly larger than in the PBS control, although they were used atthe same concentration, based on total volumes of PBS and completeblood. After incubation, however, the peptides were analyzed only in theplasma after removing the blood cells. This may be regarded as anindication that the D-Cys derivatives are distributed mainly in theplasma but are unable to penetrate or bind blood cells in a significantmanner.

2.4 Stability of uPA Peptides Against Plasmin

The peptide lead structure cyclo[21,29](D-Cys²¹Cys²⁹]-uPA₂₁₋₃₀ andmodifications thereof at position 23,cyclo[21,29][D-Cys²¹Orn²¹Cys²⁹]-uPA₂₁₋₃₀ (ornithine),cyclo[21,29][D-Cys²¹Dab²³Cys²⁹]-uPA₂₁₋₃₀ (2,4-diaminobutyric acid),cyclo[21,29][D-Cys²¹Dap²³Cys²⁹ ]-uPA₂₁₋₃₀(2,3-diaminopropionic acid),cyclo[21,29][D-Cys²¹Nle²³Cys²⁹]-uPA₂₁₋₃₀ (norleucine) andcyclo[21,29][D-Cys²¹Arg²³Cys²⁹]-uPA₂₁₋₃₀ (arginine) were tested for thesensitivity to attack by the tissue protease plasmin. The lead structurecyclo[21,29][D-Cys²¹Cys²⁹]-uPA₂₁₋₃₀ contains plasmin cleavage site knownfrom urokinase, i.e. the peptide bond between Lys²³ and Tyr²⁴.

FIG. 5 depicts HPLC profiles of peptide variants prior to (top profiles)and after (bottom profiles) incubation with plasmin. Part (A) of thefigure depicts the unaltered lead structurecyclo[21,29][D-Cys²¹Cys²⁹]-uPA₂₁₋₃₀ with lysine at position 23, (B) theornithine-substituted variant cyclo[21,29][D-Cys²¹Orn²³Cys²⁹]-uPA₂₁₋₃₀,(C) the diaminobutyric acid-substituted variantcyclo[21,29][D-Cys²¹Dab²³Cys²⁹]-uPA₂₁₋₃₀, (D) the diaminopropionicacid-substituted variant cyclo[21,29][D-Cys²¹Dap²³Cys²⁹]-uPA₂₁₋₃₀, (E)the norleucine-substituted variantcyclo[21,29][D-Cys²¹Nle²³Cys²⁹]-uPA₂₁₋₃₀ and (F) thearginine-substituted variant cyclo[21,29][D-Cys²¹Arg²³Cys²⁹]-uPA₂₁₋₃₀.The protease plasmin appears in the bottom profiles in each case atapprox. 17.5 min.

After the incubation of cyclo[21,29][D-Cys²¹Cys²⁹]-uPA₂₁₋₃₀ with plasminand HPLC analysis of the products, a new unknown peak appeared inaddition to the peak of the unaltered lead structure. The sum of bothpeak integrals corresponded to 93.5% of the peak integral of theunaltered lead structure. Accordingly, the new peak was very likely theplasmin cleavage product of cyclo[21,29][D-Cys²¹Cys²⁹]-uPA₂₁₋₃₀. Of allthe peptides which were modified at position 23 and were active withrespect to competing with uPA for binding to uPAR, the diaminobutyricacid-, ornithine- and norleucine-substituted peptides proved stableagainst plasmin (FIG. 5). In the case of the Arg²³-substituted variant anew peak appeared after incubation with plasmin, whose retention timewas nearly identical to that of the plasmin metabolite of the unalteredlead structure, i.e. the Arg²³-substituted variant is plasmin-sensitive.When exposing the diaminopropionic acid-substituted variant to plasmin,two small unidentified peaks appeared at approx. 21 min, whose retentiontime differs greatly from the retention times of the plasmin metabolitesof the lead structure and those of the Arg²³ variant. Correspondingly,it is questionable whether the small 21 min peaks indeed representspecific plasmin cleavage products of the Dap²³ variant.

Substitution of lysine at position 23 of, thecyclo[21,29][D-Cys²¹Cys²⁹]-uPA₂₁₋₃₀ lead structure by non-proteinogenicamino acids may generate stability against the tissue protease plasmin,without substantially altering the biological activity.

2.5 Anti-Angiogenetic Effectiveness

Thorax aortae were obtained from 1- to 2-month-old Wistar rats andimmediately transferred into a culture dish containing serum-free medium(RPMI). The tissue surrounding the aorta was carefully removed. Aortarings of 1 mm in length were prepared and thoroughly washed withserum-free medium. Before embedding the aorta rings in Matrigel, thebottom of each well was coated with 80 μl of gel solution. After gelformation, the aorta rings were transferred into the well, positionedand fixed by overlaying with 70 μl of gel solution. After gel formation,various amounts of the particular test peptide were introduced into thewells. Controls studied were medium alone, medium with growthsupplements and medium with growth supplements and control peptide. Thecultures were kept at 35° C. for 5 days and then studied.

The test peptides were cyclo[21,29][D-Cys²¹,Cys²⁹]-uPA₂₁₋₃₀ andcyclo[21,29][D-Cys²¹,Nle²³Cys29]-uPA₂₁₋₃₀ in dosages of 0.001, 0.1, 1,10, 25 and 50 μg/ml. The test peptides displayed distinctanti-angiogenetic actions in the in-vitro tissue culture assay.Capillary formation was found to be inhibited in the concentration rangeof about 1 μg/ml and higher. Capillary budding, i.e. the number andlength of newly formed capillaries, could be reduced at a peptideconcentration of 30 μg/ml by a factor of about 3 to 4, compared withinactive control peptides.

1. A method of inhibiting angiogenesis which comprises administering toa subject in: need thereof an effective amount of a compound of formula1

wherein X²¹—X³⁰ are monomeric building blocks, preferablyaminocarboxylic acid residues and are derived from a structure in whichX²¹=D-Cys, X²²=Asn, X²³ =Lys, X²⁴=Tyr, X²⁵=Phe, X²⁶=Ser, X²⁷=Asn,X²⁸=Ile, X²⁹=Cys and X³⁰=Trp, Y is a spacer and m is 0 or 1, and themonomeric building blocks are linked via —CONR¹ or —NR¹CO bonds, inwhich R¹ in each case independently is hydrogen, methyl or ethyl, andpharmaceutically acceptable salts and derivatives thereof, with theproviso that at least one of the amino acid residues X²¹—X³⁰ is replacedby one of the amino acid residues listed below: X²¹: Asp, Glu,2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab),D-penicillanine (D-Pen), allylglycine (Alg), ornithine (Om), Lys; X²²:Asp, Glu; X²³: Dab, Dap, His, citrulline (Cit), homocitrulline (Hci),norleucine (Nle); X²⁴: homophenylalanine (Hph),1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), thienylalanine(Thi), Trp, phenylglycin (Phg), 1-naphthylalanine (1-Nal),2-naphthylalanine (2-Nal), Cha (cyclohexylalanine); X²⁵: Trp, Tic, Thi,Hph, Phg; X²⁶: Val; X²⁷: Asp, Glu; X²⁸: Cha, 2-aminobutyric acid (Abu),tert-leucine (Tle), α-aminoisobutyric acid (Aib); and X²⁹: Asp, Glu,Dap, Dab, Alg, D-Pen, Orn, Lys.
 2. The method of claim 1, wherein atleast one of the amino acid residues X²¹—X³⁰ has one of the meaningslisted below: X²¹: D-Pen; X²³: Dap, Dab, Cit, Hci, Nle, His; X²⁴: Thi,Hph, Phg, 1-Nal, 2-Nal, Cha; X²⁵: Thi; X²⁷: Asp; X²⁸: Val, Cha.
 3. Themethod of claim 1, wherein at least one of the amino acid residuesX²¹—X³⁰ has one of the meanings listed below: X²¹: D-Pen; X²³ Dab, Nle,Cit, Hci; X²⁴: 1-Nal, 2-Nal, Cha; X²⁵: Thi; X²⁸: Cha.
 4. The method ofclaim 1, wherein X²¹—X³⁰ are monomeric building blocks, preferablyaminocarboxylic acid residues and are derived from a structure in whichX²¹=D-Cys, X²²=Asn, X²³=Dap, Dab or Nle, X²⁴=Tyr, X²⁵=Phe, X²⁶=Ser,X²⁷=Asn, X²⁸=Ile, X²⁹=Cys and X³⁰=Trp, Y is a spacer and m is 0 or 1,and the monomeric building blocks are linked via —CONR¹ or —NR¹CO bonds,in which R¹ in each case independently is hydrogen, methyl or ethyl, andpharmaceutically acceptable salts and derivatives thereof.
 5. The methodof claim 1, wherein no more than 6 of the amino acid residues X²², X²³,X²⁴, X²⁵, X²⁶, X²⁷, X²⁸ and X³⁰ are replaced with the amino acidresidues as defined in claim
 1. 6. The method of claim 1, wherein nomore than 2 of the amino acid residues X²⁴, X²⁵, X²⁸ and X³⁰ arereplaced with the amino acid residues as defined in claim
 1. 7. Themethod of claim 1, wherein angiogenesis is inhibited in cells expressinga urokinase receptor (uPAR).
 8. The method of claim 2, whereinangiogenesis is inhibited in cells expressing a urokinase receptor(uPAR).
 9. The method of claim 3, wherein angiogenesis is inhibited incells expressing a urokinase receptor (uPAR).
 10. The method of claim 4,wherein angiogenesis is inhibited in cells expressing a urokinasereceptor (uPAR).
 11. The method of claim 5, wherein angiogenesis isinhibited in cells expressing a urokinase receptor (uPAR).
 12. Themethod of claim 6, wherein angiogenesis is inhibited in cells expressinga urokinase receptor (uPAR).
 13. The use of a compound of the formula(I)

wherein X²¹—X³⁰ are monomeric building blocks, preferablyaminocarboxylic acid residues and are derived from a structure in whichX²¹=D-cys, X²²=ASN, X²³=Lys, X²⁴=Tyr, X²⁵=Phe, X²⁶−Ser, X²⁷−Asn,X²⁸=Ile, X²⁹=Cys and X³⁰=Trp, Y is a spacer and m is 0 or 1, monomericbuilding blocks are linked via —CONR¹ or —NR¹CO bonds, in which R¹ ineach case independently is hydrogen, methyl or ethyl, andpharmaceutically acceptable salts and derivatives thereof, which theproviso that at least one of the amino acid residues X²¹—X³⁰ is replacedby one of the amino acid residues listed below: X²¹: Asp, Glu,2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab),D-penicillanine (D-Pen), allylglycine (Alg), ornithine (Orn), Lys; X²²:Asp, Glu; X²³: Dab, Dap, His, citrulline (Cit), homocitrulline (Hci),norleucine (Nle); X²⁴: homophenylalanine (Hph),1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), thienylalanine(Thi), Trp, phenylglycin (Phg), 1-naphthylalanine (1-Nal),2-naphthylalanine (2-Nal), Cha (cyclohexylalanine); X²⁵: Trp, Tic, Thi,Hph, Phg; X²⁶: Val; X²⁷: Asp, Glu; X²⁸: Cha, 2-aminobutyric acid (Abu),tert-leucine (Tle), α-aminoisobutyric acid (Aib); X²⁹: Asp, Glu, Dap,Dab, Alg, D-Pen, Orn, Lys for preparing a targeting vehicle for cellsexpressing a urokinase receptor (uPAR).
 14. The use of claim 13, whereinat least one of the amino acid residues X²¹—X³⁰ has one of the meaningslisted below: X²¹: D-Pen; X²³: Dap, Dab, Cit, Hci, Nle, His; X²⁴: Thi,Hph, Phg, 1-Nal, 2-Nal, Cha; X²⁵: Thi; X²⁷: Asp; X²⁸: Val, Cha.
 15. Theuse of claim 13, wherein at least one of the amino acid residues X²¹—X³⁰has one of the meanings listed below: X²¹: D-Pen; X²³: Dab, Nle, Cit,Hci; X²⁴: 1-Nal, 2-Nal, Cha; X²⁵: Thi; X²⁸: Cha.
 16. The use of claim13, wherein X²¹—X³⁰ are monomeric building blocks, preferablyaminocarboxylic acid residues and are derived from a structure in whichX²¹=D-Cys, X²²=Asn, X²³=Dap, Dab or Nle, X²⁴=Tyr, X²⁵=Phe, X²⁶=Ser,X²⁷=ASN, X²⁸=Ile, X²⁹=Cys and X³⁰=Trp, Y is a spacer and m is 0 or 1,and the monomeric building blocks are liked via —CONR¹ or —NR¹CO bonds,in which R¹ in each case independently is hydrogen, methyl or ethyl, andpharmaceutically acceptable salts and derivatives thereof.
 17. The useof claim 13, wherein no more than 6 of the amino acid residues X²², X²³,X²⁴, X²⁵, X²⁶, X²⁷, X²⁸ and X³⁰ are replaced with the amino acidresidues as defined in claim
 13. 18. The use of claim 13, wherein nomore than 2 of the amino acid residues X²⁴, X²⁵, X²⁸ and X³⁰ arereplaced with the amino acid residues as defined in claim 13.